Simplified engineering design towards a competitive lipid-rich effluents valorization

Medium- and long-chain fatty acids and glycerol contained in the oily fraction of many food-industry effluents are excellent candidates to produce biobased high-value triacylglycerides (TAGs) and polyhydroxyalkanoates (PHAs). The typical process configuration for TAGs recovery from lipid-rich streams always includes two steps (culture enrichment plus storage compounds accumulation) whereas, for PHAs production, an additional pretreatment of the substrate for the obtainment of soluble volatile fatty acids (VFAs) is required. To simplify the process, substrate hydrolysis, culture enrichment, and accumulation (TAG and PHA storage) were coupled here in a single sequencing batch reactor (SBR) operated under the double growth limitation strategy (DGL) and fed in pulses with industrial waste fish oil during the whole feast phase. When the SBR was operated in 12 h cycles, it was reached up to 51 wt % biopolymers after only 6 h of feast (TAG:PHA ratio of 50:51; 0.423 CmmolBIOP/ CmmolS). Daily storage compound production was observed to be over 25% higher than the reached when enrichment and accumulation stages were carried in separate operational units. Increasing the feast phase length from 6 to 12 h (18 h cycle) negatively affected the DGL strategy performance and hence system storage capacity, which was recovered after also extending the famine phase in the same proportion (24 h cycle). Besides, the carbon influx during the feast phase was identified as a key operational parameter controlling storage compounds production and, together with the C/N ratio, culture selection. The different cycle configurations tested clearly modulated the total fungal abundances without no significant differences in the size of the bacterial populations. Several PHA and TAG producers were found in the mixed culture although the PHA and TAG productions were poorly associated with the increased relative abundances (RAs) of specific operational taxonomic units (OTUs).Spanish Government (AEI, Spain) through the TREASURE project CTQ 2017-83225-C2-1-R operative program FSE Galicia 2014-2020European Commissio

Rapid start-up and stable maintenance of the mainstream nitritation process based on the accumulation of free nitrous acid in a pilot-scale two-stage nitritation-anammox system

This work has been financed by the European Commission (EU) through the LIFE project ZERO WASTE WATER (LIFE19 ENV/ES/000631), the Waterworks 2014 Cofounded Call (Water JPI/Horizon) through the Pioneer_STP (PCIN-2015-022 MINECO (AEI)/ID 199 (UE)) and by the Spanish Government (AEI) through GRANDSEA (CTM2014-55397-JIN), TREASURE (CTQ2017-83225-C2-1-R) and ECOPOLYVER (PID2020-112550RB-C21 and PID2020-112550RB-C22) projects. Alba Pedrouso also acknowledges the Xunta de Galicia (Spain) for her post-doctoral fellowship (ED481B-2021-041). Authors from the USC belong to the Galician Competitive Research Group (GRC D431C-2021/37). Authors gratefully acknowledge the staff of the WWTP for their support.Two-stage partial nitritation (PN) and anammox (AMX) systems showed promising results for applying auto-trophic nitrogen removal under mainstream conditions. In this study, a pilot-scale (600 L per reactor) two-stage PN/AMX system was installed in a municipal wastewater treatment plant (WWTP) provided with a high-rate activated sludge (HRAS) system for organic carbon removal. The PN/AMX system was operated without tem-perature control (ranging from 11 to 28 degrees C) and was subjected to the same variations in wastewater charac-teristics as the WWTP (22 to 63 mg NH4+- N/L). The developed strategy is simple, does not require the addition of chemicals and is characterised by short start-up periods. The PN process was established by applying a high hydraulic load and maintained by in situ accumulated free nitrous acid (FNA) of 0.015-0.2 mg HNO2-N/L. Based on pH value, a controlled aeration strategy was applied to achieve the target nitrite to ammonium ratio in the effluent (1.1 g NO2--N/g NH4+-N) to feed the AMX reactor. Although NOB were not fully washed out from the system, nitrite accumulation remained (>99 %) stable with no evidence of NOB activity. In the AMX reactor, an overall nitrogen removal efficiency of 80 % was achieved. Regarding effluent quality, 12 +/- 3 mg TN/L was obtained, but 5 mg NO3--N/L was already in the HRAS effluent. The relative abundance of NOB showed a strong negative correlation with the FNA concentration, providing a good strategy for establishing PN under main-stream conditions.European Commission (EU) through the LIFE project ZERO WASTE WATER LIFE19 ENV/ES/000631Waterworks 2014 Cofounded Call (Water JPI/Horizon) through the Pioneer_STP (MINECO (AEI)/(UE)) ED481B-2021-041Spanish Government (AEI) 199, CTM2014-55397-JIN, CTQ2017-83225-C2-1-R, PID2020-112550RB-C21Xunta de Galicia PID2020-112550RB-C22PCIN-2015-02

Flow Cytometric Investigation of Salinicola halophilus S28 Physiological Response Provides Solid Evidence for Its Uncommon and High Ability to Face Salt-Stress Conditions

In a previous work, some bacterial strains isolated from the Saline di Tarquinia marine salterns (Viterbo, Italy) showed very unusual growth profiles in relation to temperature and salinity variations when grown in solid media. In particular, Salinicola halophilus S28 showed optimal or suboptimal growth in a very wide range of NaCl concentrations, suggesting a great coping ability with salinity variations. These intriguing outcomes did not fit with the general Salinicola halophilus description as a moderately halophilic species. Therefore, this study profiles the actual physiological status of S28 cells subjected to different NaCl concentrations to provide evidence for the actual coping ability of strain S28 with broad salinity variations. Flow cytometry was selected as the evaluation method to study the physiological status of bacterial cells subjected to different salinity levels, monitoring the strain response at different growth phases over 72 h. Strain S28 showed maximal growth at 8% NaCl; however, it grew very well with no statistically significant differences at all salinity conditions (4–24% NaCl). Flow cytometric results provided clear evidence of its actual and strong ability to face increasing salinity, revealing a good physiological response up to 24% of NaCl. In addition, strain S28 showed very similar cell physiological status at all salinity levels, as also indicated by the flat growth profile revealed in the range of 4–24% NaCl. This is the first study regarding the physiological response during the growth of halophilic bacteria under different conditions of salinity via flow cytometry. This technique represents an effective tool for the investigation of the physiological status of each cell, even if it is somehow underrated and underused by microbiologists for this purpose

Impact on the microbial population during biological volatile fatty acid production from olive mill solid waste

This work was funded by the Spanish Ministry of Economy, Industry and Competitiveness (Ref. PID2020-116698RB-100). Dr. Antonio Serrano was funded by the Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Junta de Andalucía - EMERGIA20_00114). Juan Cubero-Cardoso was funded by the Recualificación del Profesorado Universitario (Next Generation European Funds and Spanish University Ministry system).Supplementary material: Download Word document (34KB), 1-s2.0-S2352186423004054-mmc1Volatile fatty acids (VFAs) revalorisation from waste products are key in achieving industrial sustainability and circular economic goals. Hence, the objective of this work was to correlate the adaptability of the microbial community in olive mill solid waste (OMSW) anaerobic fermentation processes, to the production of VFAs under different pH conditions, i.e. under acidic (pH 4 &5), neutral (pH 6 & 7) and alkaline conditions (pH 9 & 10). At neutral conditions, anaerobic digestion exhibited minimal accumulation of VFAs, as they were primarily biotransformed to methane, where no significant changes in the microbial community were observed. At acidic conditions, a diverse profile of VFAs were present in the reactors, although the VFA production was limited to around 20 % of fed OMSW. Despite the low accumulation, the VFA profile at pH 5 was more complex than those at alkaline conditions, accounting propionic acid as the main VFA compound produced at pH 5 (60 % of the total VFAs). Acidic conditions entailed a shift in the microbial composition compared to the initial inoculum, although the reactors maintained similar diversity indices. At alkaline conditions, around 50 % of the fed OMSW was accumulated as VFAs, mainly as acetic acid. Overall, a lower diversity and higher dominance corresponded to a less diverse VFAs profile, such as the preponderance of acetic acid correlated with a microbial diversity decrease and the increased dominance of Tissirella.Spanish Ministry of Economy, Industry and Competitiveness (Ref. PID2020-116698RB-100)Junta de Andalucía - EMERGIA20_00114Recualificación del Profesorado Universitario (Next Generation European Funds and Spanish University Ministry system

Dynamics of PHA-Accumulating Bacterial Communities Fed with Lipid-Rich Liquid Effluents from Fish-Canning Industries

This research was supported by the Spanish Government (Spanish Ministry of Science and Innovation) through the TREASURE-MICROSALT (CTQ2017-83225-C2-2-R) and ECOPOLYVER-MACROPOLYVER (PID2020-112550RC22) projects.The biosynthesis of polyhydroxyalkanoates (PHAs) from industrial wastes by mixed microbial cultures (MMCs) enriched in PHA-accumulating bacteria is a promising technology to replace petroleum-based plastics. However, the populations’ dynamics in the PHA-accumulating MMCs are not well known. Therefore, the main objective of this study was to address the shifts in the size and structure of the bacterial communities in two lab-scale sequencing batch reactors (SBRs) fed with fish-canning effluents and operated under non-saline (SBR-N, 0.5 g NaCl/L) or saline (SBR-S, 10 g NaCl/L) conditions, by using a combination of quantitative PCR and Illumina sequencing of bacterial 16S rRNA genes. A double growth limitation (DGL) strategy, in which nitrogen availability was limited and uncoupled to carbon addition, strongly modulated the relative abundances of the PHA-accumulating bacteria, leading to an increase in the accumulation of PHAs, independently of the saline conditions (average 9.04 wt% and 11.69 wt%, maximum yields 22.03 wt% and 26.33% SBR-N and SBR-S, respectively). On the other hand, no correlations were found among the PHAs accumulation yields and the absolute abundances of total Bacteria, which decreased through time in the SBR-N and did not present statistical differences in the SBR-S. Acinetobacter, Calothrix, Dyella, Flavobacterium, Novosphingobium, Qipengyuania, and Tsukamurella were key PHA-accumulating genera in both SBRs under the DGL strategy, which was revealed as a successful tool to obtain a PHA-enriched MMC using fish-canning effluents.Spanish Government (Spanish Ministry of Science and Innovation) CTQ2017-83225-C2-2-R PID2020-112550RC2

Structure of fungal communities in sequencing batch reactors operated at different salinities for the selection of triacylglyceride-producers from a fish-canning lipid-rich waste stream

Oleaginous fungi natively accumulate large amounts of triacylglycerides (TAG), widely used as precursors for sustainable biodiesel production. However, little attention has been paid to the diversity and roles of fungal mixed microbial cultures (MMCs) in sequencing batch reactors (SBR). In this study, a lipid-rich stream produced in the fish-canning industry was used as a substrate in two laboratory-scale SBRs operated under the feast/famine (F/F) regime to enrich microorganisms with high TAG-storage ability, under two different concentrations of NaCl (SBR-N: 0.5 g/L; SBR-S: 10 g/L). The size of the fungal community in the enriched activated sludge (EAS) was analyzed using 18S rRNA-based qPCR, and the fungal community structure was determined by Illumina sequencing. The different selective pressures (feeding strategy and control of pH) implemented in the enrichment SBRs throughout operation increased the abundance of total fungi. In general, there was an enrichment of genera previously identified as TAG-accumulating fungi (Apiotrichum, Candida, Cutaneotrichosporon, Geotrichum, Haglerozyma, Metarhizium, Mortierella, Saccharomycopsis, and Yarrowia) in both SBRs. However, the observed increase of their relative abundances throughout operation was not significantly linked to a higher TAG accumulation.Spanish Government CTQ2017 - 83225-C2 - 1- R CTQ2017 - 83225-C2 - 2-R PID-2020 - 112550RB-C2

Revealing the dissimilar structure of microbial communities in different WWTPs that treat fish-canning wastewater with different NaCl content

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research was supported by the Spanish Government (Spanish Ministry of Science and Innovation) through TREASURE-TECHNOSALT (CTQ2017-83225-C2-1-R) and TREASURE-MICROSALT (CTQ2017-83225-C2-2-R) projects. The authors Alba Roibas-Rozas and Anuska Mosquera-Corral belong to the Galician Competitive Research Group GRC-ED431C 2017-29 and to the CRETUS Strategic Partnership (ED431E 2018/01) . All these programmes are co-funded by FEDER (UE) . Funding for open access charge: Universidad de Granada/CBUA.Studies that characterize the microbial communities in wastewater treatment plants (WWTPs) are numerous, yet similar studies in industrial WWTPs treating fish-canning effluents are limited. The microbial communities in samples of 4 fish-canning WWTPs that operated under different NaCl concentrations were investigated by qPCR and partial 16S rRNA gene Illumina sequencing. The absolute abundances of key microbial populations (Total Bacteria, Archaea and Fungi, ammonium oxidizing bacteria (AOB), Mycolata, Candidatus Microthrix, Ca. Accumulibacter and Ca. Competibacter) presented statistical differences among the WWTPs. The NaCl concentration negatively affected the absolute abundance of Bacteria and Fungi, filamentous, and phosphate (PAO) and glycogen (GAO) accumulating bacteria, while AOB and Ca. Microthrix populations were statistically higher in the WWTP with higher NaCl contents. On the other hand, the main bacterial operational taxonomic units (OTUs) were classified as members of Kouleothrix (Chloroflexia, Chloroflexi) and Tetrasphaera (Actinomycetia, Actinobacteria), family Beijerinckiaceae (Alphaproteobacteria, Proteobacteria), order Betaproteobacteriales (Gammaproteobacteria, Proteobacteria), Sphingobacteriales (Sphingobacteriia, Bacteroidetes) and Frankiales (Actinobacteria, Actinobacteria), class Anaerolineae (Chloroflexi), phylum Chloroflexi and Bacteria_unclassified. The structure of the bacterial community was highly dissimilar among the 4 WWTPs, as the identities of the dominant OTUs differed significantly among them. Therefore, the individual characteristics of the different WWTPs, mainly NaCl concentration, were responsible for the narrow assemblage of the bacterial communities. Different OTUs belonging to the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes and Proteobacteria were revealed as salttolerant. Taking into account these results, NaCl content was an important driver of the abundance of microbial populations and the bacterial community structure in the analysed industrial facilities.Spanish Government (Spanish Ministry of Science and Innovation) CTQ2017-83225-C2-1-R CTQ2017-83225-C2-2-REuropean CommissionGalician Competitive Research Group GRC-ED431C 2017-29CRETUS Strategic Partnership ED431E 2018/0

Bacterial Communities in the Rhizosphere of Amilaceous Maize (Zea mays L.) as Assessed by Pyrosequencing

Maize (Zea mays L.) is the staple diet of the native peasants in the Quechua region of the Peruvian Andes who continue growing it in small plots called chacras following ancestral traditions. The abundance and structure of bacterial communities associated with the roots of amilaceous maize has not been studied in Andean chacras. Accordingly, the main objective of this study was to describe the rhizospheric bacterial diversity of amilaceous maize grown either in the presence or the absence of bur clover cultivated in soils from the Quechua maize belt. Three 16S rRNA gene libraries, one corresponding to sequences of bacteria from bulk soil of a chacra maintained under fallow conditions, the second from the rhizosphere of maize-cultivated soils, and the third prepared from rhizospheric soil of maize cultivated in intercropping with bur clover were examined using pyrosequencing tags spanning the V4 and V5 hypervariable regions of the gene. A total of 26031 sequences were found that grouped into 5955 distinct operational taxonomic units which distributed in 309 genera. The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from bulk soil. One hundred ninety seven genera were found in the bulk soil library and 234 and 203 were in those from the maize and maize/bur clover-cultivated soils. Sixteen out of the 309 genera had a relative abundance higher than 0.5% and the were (in decreasing order of abundance) Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis. Genera Gp4 and Gp6 of the Acidobacteria, Gemmatimonas and Rhodoferax were the most abundant in bulk soil, whereas Flavobacterium, Dechloromonas and Ohtaekwangia were the main genera in the rhizosphere of maize intercropped with bur clover, and Gp4, Subdivision3 genera incertae sedis of phylum Verrucomicrobia, Gp6 and Rhodoferax were the main genera in the rhizosphere of maize plants. Taken together, our results suggest that bur clover produces specific changes in rhizospheric bacterial diversity of amilaceous maize plants.Peer reviewe

Analysis of the denitrification pathway and greenhouse gases emissions in Bradyrhizobium sp. strains used as biofertilizers in South America

Aims Greenhouse gases are considered potential atmospheric pollutants, with agriculture being one of the main emission sources. The practice of inoculating soybean seeds with Bradyrhizobium sp. might contribute to nitrous oxide (N2O) emissions. We analyzed this capacity in five of the most used strains of Bradyrhizobium sp. in South America. Methods and Results We analyzed the denitrification pathway and N2O production by B. japonicum E109 and CPAC15, B. diazoefficiens CPAC7, and B. elkanii SEMIA 587 and SEMIA 5019, both in free‐living conditions and symbiosis with soybean. The in silico analysis indicated the absence of nosZ genes in B. japonicum and the presence of all denitrification genes in B. diazoefficiens strains, as well as the absence of nirK, norC and nosZ genes in B. elkanii. The in planta analysis confirmed the N2O production under saprophytic conditions or symbiosis with soybean roots nodules. In the last case up to 26·1 and 18·4 times higher in plants inoculated with SEMIA5019 and E109 respectively, than in those inoculated with USDA110. Conclusions The strains E109, SEMIA 5019, CPAC15 and SEMIA 587 showed the highest N2O production both as free‐living cells and in symbiotic conditions in comparison with USDA110 and CPAC7, which do have the nosZ gene. Although norC and nosZ could not be identified in silico or in vitro in SEMIA 587 and SEMIA 5019, these strains showed capacity to produce N2O in our experimental conditions. Significance and Impact of Study This is the first report to analyze and confirm the incomplete denitrification capacity and N2O production in four of the five most used strains of Bradyrhizobium sp. for soybean inoculation in South America.Fil: Obando Castellanos, Dolly Melissa. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Correa-Galeote, David. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Castellano-Hinojosa, Antonio. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Gualpa, José Luis. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-microorganismo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hidalgo, Alba. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: de Dios Alché, Juan. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Bedmar, Eulogio. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentin

Rapid start-up and stable maintenance of the mainstream nitritation process based on the accumulation of free nitrous acid in a pilot-scale two-stage nitritation-anammox system

Two-stage partial nitritation (PN) and anammox (AMX) systems showed promising results for applying autotrophic nitrogen removal under mainstream conditions. In this study, a pilot-scale (600 L per reactor) two-stage PN/AMX system was installed in a municipal wastewater treatment plant (WWTP) provided with a high-rate activated sludge (HRAS) system for organic carbon removal. The PN/AMX system was operated without temperature control (ranging from 11 to 28 °C) and was subjected to the same variations in wastewater characteristics as the WWTP (22 to 63 mg NH4+- N/L). The developed strategy is simple, does not require the addition of chemicals and is characterised by short start-up periods. The PN process was established by applying a high hydraulic load and maintained by in situ accumulated free nitrous acid (FNA) of 0.015–0.2 mg HNO2-N/L. Based on pH value, a controlled aeration strategy was applied to achieve the target nitrite to ammonium ratio in the effluent (1.1 g NO2–-N/g NH4+-N) to feed the AMX reactor. Although NOB were not fully washed out from the system, nitrite accumulation remained (>99 %) stable with no evidence of NOB activity. In the AMX reactor, an overall nitrogen removal efficiency of 80 % was achieved. Regarding effluent quality, 12 ± 3 mg TN/L was obtained, but 5 mg NO3–-N/L was already in the HRAS effluent. The relative abundance of NOB showed a strong negative correlation with the FNA concentration, providing a good strategy for establishing PN under mainstream conditionsThis work has been financed by the European Commission (EU) through the LIFE project ZERO WASTE WATER (LIFE19 ENV/ES/000631), the Waterworks 2014 Cofounded Call (Water JPI/Horizon) through the Pioneer_STP (PCIN-2015-022 MINECO(AEI)/ID 199 (UE)) and by the Spanish Government (AEI) through GRANDSEA (CTM2014-55397-JIN), TREASURE (CTQ2017-83225-C2-1-R) and ECOPOLYVER (PID2020-112550RB-C21 and PID2020-112550RB-C22) projects. Alba Pedrouso also acknowledges the Xunta de Galicia (Spain) for her postdoctoral fellowship (ED481B-2021-041). Authors from the USC belong to the Galician Competitive Research Group (GRC D431C-2021/37)S